TETRAHYDRO-beta-CARBOLINE DERIVATIVES, SYNTHESIS AND USE THEREOF

ABSTRACT

Certain 2-halophenyl, 2,4-dihalophenyl (e.g. 2,4-dichlorophenyl), 3,4-dichlorophenyl (e.g. 3,4-dichlorophenyl), 2,6-dichlorophenyl (2,6-dichlorophenyl) and 2,5-diakoxyphenyl (e.g. 2,5-dimethoxyphenyl) derivatives of tetrahydro-β-carbolines are provided, along with their pharmaceutically acceptable salts; prodrugs and solvates, and compositions containing the compounds. The compounds are useful for the prevention and treatment of cancer, and other indications where PDE5 inhibitors have shown benefits including erectile dysfunction, pulmonary hypertension, enhancing cognitive function, cystic fibrosis, or enhancing the activity of conventional chemotherapeutic drugs. Methods for fabricating the compounds are also provided.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was partially supported by a grant No. 5R01CA131378 fromNational Institutes of Health, National Cancer Institute and the USGovernment has certain rights in the invention.

TECHNICAL FIELD

The present disclosure relates to derivatives of tetrahydro-β-carbolinesand in particular 2-halophenyl, 2,4-dihalophenyl, 3,4-dihalophenyl,2,6-dihalophenyl and 2,5-dialkoxyphenyl derivatives oftetrahydro-β-carbolines including 2-chlorophenyl, 2-bromophenyl,2,4-dichlorophenyl, 3,4-dichlorophenyl, 2,6-dichlorophenyl and2,5-dimethoxyphenyl derivatives of tetrahydro-β-carbolines. The presentdisclosure also relates to pharmaceutical compositions comprising thedisclosed derivatives of tetrahydro-β-carbolines, as well as a method ofusing the compounds for the prevention or treatment of cancer in amammal. In addition, the disclosed compounds are for other indicationswhere PDE5 inhibitors have shown benefits including erectiledysfunction, pulmonary hypertension, enhancing cognitive function,cystic fibrosis, or enhancing the activity of conventionalchemotherapeutic drugs. The present disclosure also relates to a methodfor producing the disclosed compounds.

Background of Disclosure

Phosphodiesterase type 5 (PDE5) is an important enzyme involved inregulating intracellular cyclic guanosine monophosphate (cGMP)signaling. PDE5 catalyzes the hydrolysis of cGMP into 5′ guanosinemonophosphate, whereby inhibition leads to increased magnitude orduration of the cGMP signal. PDE5 is an essential regulator of normalphysiological processes, such as smooth muscle contraction andrelaxation and may also play an important role in a variety ofpathological conditions including pulmonary hypertension, cognitivefunction, cystic fibrosis, and cancer. For example, PDE5 is a major cGMPdegrading PDE isozyme in penile corpus cavernosum tissue, whereby itsinhibition by drugs such as sildenafil, vardenafil, or tadalafil canenhance penile erection upon sexual arousal.^(1, 2) Inhibition of PDE5may also benefit patients with pulmonary hypertension or cysticfibrosis, while additional indications are being studied including theenhancement of cognitive function, increasing efficacy of conventionalchemotherapy, as well as being cardioprotective.

Recent studies have shown that PDE5 and possibly additional cGMP-PDEsare expressed in various carcinomas such as those derived from thecolon, breast, lung and bladder. Moreover, cGMP PDE inhibition andconsequent high intracellular levels of cGMP may be associated with theapoptotic activities of certain drugs.^(4, 5) For example, exisulind andits analogs (CP78, CP461, CP248) have been reported to selectivelyinduce apoptosis of tumor cells derived from a variety of cancersincluding colon, bladder, prostate, breast and lung. Exisulind and itsanalogs maintained similar rank order of potency to induce apoptosis andinhibit tumor cell growth compared with cGMP PDE inhibition. Suchcompounds also caused a sustained elevation of intracellular cGMP levelsin colon tumor cells. Therefore, it is proposed that cGMP mediates themechanism underlying the apoptosis inducing properties of exisulind inneoplastic cells.⁴⁻⁷ These effects in neoplastic cells appear to be notsolely dependent on the specific inhibition of PDE5, but rather, arerelated to inhibition of additional cGMP-PDEs. Previous studiestherefore indicate that it is important to inhibit multiple cGMP PDEisoforms, although the exact isozymes involved have not been welldefined.⁷ Accordingly, highly selective and potent PDE5 inhibitors suchas tadalafil would not be expected to have anticancer properties.Additionally, there may be other reasons why conventional PDE5inhibitors do not have anticancer activity that have not yet beendelineated.

Even though significant advances have occurred in the treatment ofcancer, it still remains a major worldwide health concern.Notwithstanding the advances in treatments for cancer and otherhyperproliferative diseases there still remains room for improved drugsthat are effective with minimal toxicity.

SUMMARY OF DISCLOSURE

The present disclosure relates to compounds represented by the followingformulae (1) and (2):

wherein in formula (1), R₁ is a hydroxy or an alkoxy group containing1-4 carbon atoms; R₂ is an acyl halo group containing 2-3 carbon atoms;R₃ is a halo group or an alkoxy group containing 1-4 carbon atoms and R₄is H when R₃ is halo and is an alkoxy group containing 1-4 carbon atomswhen R₃ is an alkoxy group containing 1-4 carbon atoms; pharmaceuticallyacceptable salt thereof; prodrugs thereof or solvates thereof; and

wherein in formula (2), R₁ is a hydroxy or an alkoxy group containing1-4 carbon atoms; R₂ is an acyl halo group containing 2-3 carbon atoms;R₃ is a halo group and R₄ is H or halo; pharmaceutically acceptable saltthereof; prodrugs thereof or solvates thereof.

Another aspect of the present disclosure relates to pharmaceuticalcompositions containing a compound represented by formula (1) and/orformula (2); pharmaceutically acceptable salt thereof; prodrug thereofor solvate thereof.

A still further aspect of the present invention relates to a method forpreventing or treating a mammalian host at risk of developing cancer orhas been diagnosed with cancer, which comprises administering to saidhost an effective amount of at least one compound represented by theformula (1) or formula (2) or both; a pharmaceutically acceptable saltthereof; a prodrug thereof or a solvate thereof; alone or in combinationwith known preventive or therapeutic drugs.

Another aspect of the present disclosure relates to treating men witherectile dysfunction by administering an amount effective for treatingerectile dysfunction of at least one compound represented by the formula(1) or formula (2) or both; a pharmaceutically acceptable salt thereof;a prodrug thereof or a solvate thereof; alone or in combination withknown drugs that are useful for treating erectile dysfunction.

A still further aspect of the present disclosure relates to treating amammalian host suffering from pulmonary hypertension, which comprisesadministering to the host an amount effective for treating pulmonaryhypertension of at least one compound represented by the formula (1) orformula (2) or both; a pharmaceutically acceptable salt thereof; aprodrug thereof or a solvate thereof; alone or in combination with knowndrugs that are useful for pulmonary hypertension.

The present disclosure also relates to methods for preparing compoundsaccording to formulae (1) and (2) above.

Still other objects and advantages of the present disclosure will becomereadily apparent by those skilled in the art from the following detaileddescription, wherein it is shown and described only the preferredembodiments, simply by way of illustration of the best mode. As will berealized, the disclosure is capable of other and different embodiments,and its several details are capable of modifications in various obviousrespects, without departing from the disclosure. Accordingly, thedescription is to be regarded as illustrative in nature and not asrestrictive.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-B illustrate the effect of compound 2f and tadalafil on cGMPelevation in human MDA-MB-231 breast tumor cells. FIG. 1A illustratesthe induction of intracellular cGMP levels in cultured human MDA-MB-231breast tumor cells treated for 30 minutes with compound 2f. FIG. 1Billustrates the inability of tadalafil to alter cGMP levels in humanMDA-MB-231 breast tumor cells. The + indicates the effects of a positivecontrol, sulindac sulfide tested at 100 μM. The cells were treated for30 minutes prior to measuring intracellular cGMP levels by immunoassay.The * indicates statistical significance with p<0.05.

FIG. 2 illustrates cGMP phosphodiesterase inhibitory activity ofcompound 2f. Whole cell lysates from human MDA-MB-231 breast tumor cellstreated for 18 hours with compound 2f were assayed for cGMPphosphodiesterase activity using a fluorescence polarization assay(Molecular Devices Inc).

FIGS. 3A-B illustrate the anticancer activity of compound 2j. FIG. 3Ashows in vitro tumor cell growth inhibitory activity of compound 2j inthe human Caki-1 renal tumor cell line with an IC₅₀ value of 2.3 nM. Thecells with treated for 72 hours prior to measuring cell viability usingthe Cell Titer Glo assay (Promega Corp.). FIG. 3B shows in vivo tumorgrowth inhibitory of compound 2j using the human Caki-1 renal tumormouse xenograft model. Tumor bearing mice were treated at a dose of 10mg/kg one time per day for 22 days.

BEST AND VARIOUS MODES

The present disclosure relates to compounds represented by the followingformulae (1) and (2):

wherein in formula (1), R₁ is a hydroxy or an alkoxy group containing1-4 carbon atoms; R₂ is an acyl halo group containing 2-3 carbon atoms;R₃ is a halo group or an alkoxy group containing 1-4 carbon atoms and R₄is H when R₃ is halo and is an alkoxy group containing 1-4 carbon atomswhen R₃ is an alkoxy group containing 1-4 carbon atoms; pharmaceuticallyacceptable salt thereat prodrugs thereof or solvates thereat and

wherein in formula (2), R₁ is a hydroxy or an alkoxy group containing1-4 carbon atoms; R₂ is an acyl halo group containing 2-3 carbon atoms;R₃ is a halo group and R₄ is H or halo; pharmaceutically acceptable saltthereof; prodrugs thereof or solvates thereof.

It is noted that in the interest of avoiding overlap between formula (1)and formula (2), that when R₃ is in position 2 and R₄ is in position 5,R₄ is halo and not H.

Examples of alkoxy groups include methoxy, ethoxy, propoxy and butoxy,with methoxy being most typical.

Examples of halo groups include chloro, bromo and fluoro. The mosttypical halo group employed in the R₂ acylhalo group is chloro, and themost typical halo group for R₃ is chloro or bromo and the most typicalgroups for R₄ are hydrogen, methoxy or chloro. The most typical acylhalogroup for R₂ is ethanoyl chloride.

It is of course understood that the compounds of the present disclosurerelate to all optical isomers and stereo-isomers at the various possibleatoms of the molecule, unless specified otherwise.

A “prodrug” is a compound that is converted within the body into itsactive form that has a medical effect. Prodrugs may be useful when theactive drug may be too toxic to administer systemically, the active drugis absorbed poorly by the digestive tract, or the body breaks down theactive drug before it reaches its target. Methods of making prodrugs aredisclosed in Hans Bundgaard, DESIGN OF PRODRUGS (Elsevier SciencePublishers B.V. 1985), which is incorporated herein by reference in itsentirety.

Prodrug forms of the compounds bearing various nitrogen functions(amino, hydroxyamino, hydrazino, guanidino, amidino, amide, etc.) mayinclude the following types of derivatives where each R groupindividually may be hydrogen, substituted or unsubstituted alkyl, aryl,alkenyl, alkynyl, heterocycle, alkylaryl, aralkyl, aralkenyl, aralkynyl,cycloalkyl or cycloalkenyl groups as defined above.

Carboxamides, —NHC(O)R

Carbamates, —NHC(O)OR

(Acyloxy)alkyl Carbamates, NHC(O)OROC(O)R

Enamines, —NHCR(═CHCRO₂R) or —NHCR(═CHCRONR₂)

Schiff Bases, —N═CR₂

Mannich Bases (from carboximide compounds), RCONHCH₂NR₂

Preparations of such prodrug derivatives are discussed in variousliterature sources (examples are: Alexander et al., J. Med. Chem. 1988,31, 318; Aligas-Martin et al., PCT WO pp/41531, p. 30). The nitrogenfunction converted in preparing these derivatives is one (or more) ofthe nitrogen atoms of a compound of the invention.

Prodrug forms of carboxyl-bearing compounds of the disclosure includeesters (—CO₂R) where the R group corresponds to any alcohol whoserelease in the body through enzymatic or hydrolytic processes would beat pharmaceutically acceptable levels. Another prodrug derived from acarboxylic acid form of the disclosure may be a quaternary salt type ofstructure described by Bodor et al., J. Med. Chem. 1980, 23, 469.

“Pharmaceutically acceptable salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. The compounds of this disclosure form acidand base addition salts with a wide variety of organic and inorganicacids and bases and includes the physiologically acceptable salts whichare often used in pharmaceutical chemistry. Such salts are also part ofthis disclosure. Typical inorganic acids used to form such salts includehydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric,hypophosphoric and the like. Salts derived from organic acids, such asaliphatic mono and dicarboxylic acids, phenyl substituted alkonic acids,hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphaticand aromatic sulfonic acids may also be used. Such pharmaceuticallyacceptable salts thus include acetate, phenylacetate, trifluoroacetate,acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate,naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, cabrate,caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate,heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, teraphthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzene-sulfonate, p-bromobenzenesulfonate, chlorobenzenesulfonate,ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toleunesulfonate,xylenesulfonate, tartarate, and the like.

Bases commonly used for formation of salts include ammonium hydroxideand alkali and alkaline earth metal hydroxides, carbonates, as well asaliphatic and primary, secondary and tertiary amines, aliphaticdiamines. Bases especially useful in the preparation of addition saltsinclude sodium hydroxide, potassium hydroxide, ammonium hydroxide,potassium carbonate, methylamine, diethylamine, and ethylene diamine.

“Solvates” refers to the compound formed by the interaction of a solventand a solute and includes hydrates. Solvates are usually crystallinesolid adducts containing solvent molecules within the crystal structure,in either stoichiometric or nonstoichiometric proportions.

The term “comprising” (and its grammatical variations) as used herein isused in the inclusive sense of “having” or “including” and not in theexclusive sense of “consisting only of.” The terms “a” and “the” as usedherein are understood to encompass the plural as well as the singular.

The general synthesis of the target derivatives is illustrated in thereaction schemes below:

Both D-tryptophan and L-tryptophan methyl ester were synthesized by ageneral synthetic procedure for amino acid esters.⁸ The D- andL-Tryptophan methyl esters and 2-bromobenzaldehyde,2,5-dimethoxybenzaldehyde, 2,4-dichlorobenzaldehyde,3,4-dichlorobenzaldehyde or 2,6-dichlorobenzaldehdye were subjected toPictet-Spengler reaction under non-stereospecific conditions to give thecorresponding cis- and trans-1,3-disubstituted THBCs (1a-r). It isworthy to mention that 2,6-dichlorobenzaldehdye gave only the cisisomer. See the examples presented herein below. The produced cis- andtrans-diastereomers of the 1,3-disubistituted THBC, if any wereseparated by column chromatography using CH₂Cl₂ as an eluent. Reactionof the respective THBC with chloroacetyl chloride provided thecorresponding amide derivative (2a-r). See the examples presented hereinbelow.

The cis/trans stereochemistry for the THBCs (1a-r) was assigneddepending on the comprehensive study of ¹³C NMR spectroscopy dataestablished in previous literature, the signals for C-1 in the transdiastereomers were clearly at a lower δ or ppm value in the ¹³C NMR fromthose of the corresponding cis isomers in the carbon spectrum. This isprobably due to the 1,3 interactions present in the trans-isomer. ⁹⁻¹¹

Moreover, a correlation exists between R_(f) value on TLC and thestereochemistry of the 1,3-disubstituted THBC 1a-r, where cis-isomer issystematically less polar than the trans isomer, if any ¹².

Mass spectrometry to derivatives 1a-d and 2a-d showed the molecular ionpeaks at m/z M⁺ and M⁺+2 due to the isotopic nature of bromine atom.Derivatives 1i-r and 2a-r showed molecular ion peaks at M⁺ and M⁺+2 andM⁺+4 in the ratio of 9:6:1, a pattern characteristic of dichlorocompounds. Moreover, the 1,3-disubstituted THBC derivatives 1a-r showedmolecular ion peaks that were also the base peaks indicating theirstable nature.

¹H-NMR of the amides 2a-r showed a peak at about 4.2 ppm due to theprotons of the —CH₂Cl function.

Results and Discussion

Most of compounds were evaluated for in vitro tumor cell growthinhibitory activity in 2 steps; first, the percentage inhibition at aconcentration of 100 μM was performed in triplicate, then for compoundsdisplaying a percentage of inhibition >70% were evaluated by testing arange of 10 concentrations with at least two replicates perconcentration to calculate an IC₅₀ value. The results are summarized inTable 1 and 2.

TABLE 1 Tumor cell growth inhibitory activity of1,3-disubstituted-tetrahydro-β- carbolines against human MDA-MB-231breast tumor cells. Growth % Growth Inhibition Inhibition IC₅₀, μM Cpd #Stereochemistry X, Y at 10 μM (95% CI) 1i 1R, 3R 2,4-  29 +/− 2.16 NDdichloro 1j 1S, 3R 2,4-  78 +/− 1.02 6.73 dichloro (2.21-20.5) 1k 1S, 3S2,4-  50 +/− 1.05 ND dichloro 1l 1R, 3S 2,4-  28 +/− 0.13 ND dichloro 1m1R, 3R 3,4-  43 +/− 2.41 ND dichloro 1n 1S, 3R 3,4- 100 +/− 0.02 4.62dichloro (4.09-5.23) 1o 1S, 3S 3,4-  62 +/− 0.49 6.98 dichloro(5.93-8.22) 1p 1R, 3S 3,4-  48 +/− 2.12 ND dichloro 1q 1R, 3R 2,6-  25+/− 0.07 ND dichloro 1r 1S, 3S 2,6-  52 +/− 0.40 ND dichloro

TABLE 2 Tumor cell growth inhibitory activity of1,2,3-trisubstituted-tetrahydro-β- carbolines against human MDA-MB-231breast tumor cells. Growth % Growth Inhibition Cpd Inhibition IC₅₀, μM #Stereochemistry X, Y at 10 μM (95% CI) 2i 1R, 3R 2,4- 98 +/− 0.01 0.56dichloro (0.52-0.61) 2j 1S, 3R 2,4- 98 +/− 0.07 0.07 dichloro(0.05-0.11) 2k 1S, 3S 2,4-  55 +/− 0.039 ND dichloro 2l 1R, 3S 2,4-  98+/− 0.003 1.24 dichloro (1.05-1.48) 2m 1R, 3R 3,4- 40 +/− 2.46 NDdichloro 2n 1S, 3R 3,4- 50 +/− 0.10 ND dichloro 2o 1S, 3S 3,4- 43 +/−4.95 ND dichloro 2p 1R, 3S 3,4- 55 +/− 1.07 ND dichloro 2q 1R, 3R 2,6-99 +/− 0.01 0.05 dichloro (0.04-0.05) 2r 1S, 3S 2,6- 24 +/− 0.54 0.42dichloro (0.35-0.51)

Compounds 2a-h were tested for tumor cell growth inhibitory activityusing the human breast MDA-MB-231 and ZR75-1 tumor cell lines and thecolon CACO-2, SW-1116, and HT-29 tumor cell lines. The results aresummarized in Table 3.

TABLE 3 Inhibitory effect (IC₅₀ value, μM) of example compounds on thegrowth of human breast and colon tumor cell lines. ND = Not determined.Stereo- Cmpd# Chemical structure chemistry MDA-MB-231 ZR75-1 CACO-2SW1116 HT-29 2a 2b 2c 2d

1R,3R 1S,3R 1S,3S 1R,3S 0.06 0.02 0.02 0.25 3.52 1.10 1.10 3.61 0.791.60 0.10 0.55 2.79 4.30 2.72 1.05 1.90 1.16 0.77 3.20 2e 2f 2g 2h

1R,3R 1S,3R 1S,3S 1R,3S 4.25  0.006 1.60 0.04 5.70 1.20 4.13 3.95 3.500.01 3.04 0.29   7.20   4.40   3.71 >10 3.38 0.45 6.12 5.59 2i 2j 2k 2l

1R,3R 1S,3R 1S,3S 1R,3S 0.56 0.07 0.38 1.24 ND ND ND ND ND ND ND ND NDND ND ND ND ND ND ND 2m 2n 2o 2p

1R,3R 1S,3R 1S,3S 1R,3S >10   1.2 >10 >10 ND ND ND ND ND ND ND ND ND NDND ND ND >10 ND >10 2q 2r

1R,3R 1S,3S 0.04 0.44 ND ND ND ND ND ND 1.5 1.0

From Table 3, compounds (2a-f, 2i, 2j, 2k, 2l, 2n, 2q and 2r) showedappreciable activity to inhibit the growth of human breast and colontumor cell lines with IC₅₀ ranging from 0.006-7.20 μM. Compound 2f wasthe most active in the MDA-MB-231 with an IC₅₀ value of 0.006 μM.Compound 2f was also tested in a large panel of human tumor cell linesderived from histologically diverse tumor types. As shown in Table 4 andas evident by low IC₅₀ values, compound 2f displayed potent broadspectrum growth inhibitory activity against the majority of the tumortypes that were tested, but was especially active against tumor cellsderived from hematological and renal tumors, as well asestrogen-receptor, progesterone receptor, and Her2 negative (triplenegative) breast tumor cell lines. The high potency was especiallynoteworthy in the NCI/ADR-RES tumor cell line, which is known to overexpress the drug efflux protein, P-glycoprotein (ABCB1) that mediatesresistance to multiple chemotherapeutic drugs. By contrast, normal humanmammary epithelial cells (HMEC) was found to be insensitive to compound2f, which reflects as an element of tumor selectivity and a potentialsafety advantage of the compound compared with conventionalchemotherapeutic drugs.

The mechanism of action by which these compounds inhibit tumor cellgrowth is believed to involve the inhibition of cGMP PDE isozymes thatare expressed in sensitive tumor cells. As evidence for this mechanism,the ability of compound 2f to increase intracellular cGMP levels inhuman MDA-MB-231 breast tumor (triple negative) cells at concentrationsthat inhibit tumor growth is shown in FIG. 1. The uniqueness of thiscompound compared with tadalafil, which does not inhibit tumor cellgrowth, is evident by its ability to increase cyclic GMP levels inMDA-MB-231 tumor cells, whereas tadalafil was ineffective. Consistentwith these observations, compound 2f selectively inhibited cGMP PDEactivity in lysates from MDA-MB-231 breast tumor cells as shown in FIG.2.

Compound 2j was also found to be highly potent by testing in a largepanel of human tumor cell lines as summarized in Table 5. As shown inFIG. 3, compound 2j inhibited the growth of human Caki-1 renal tumorcells with an IC₅₀ value of 0.002 μM. To demonstrate the in vivoantitumor efficacy of this class of compounds, compound 2j was evaluatedin the human Caki-1 renal tumor mouse xenograft model. As shown in FIG.3, compound 2j strongly suppressed tumor growth in this experimentalmouse model.

TABLE 4 Growth inhibitory activity of compound 2f in a panel of humantumor cell lines. Compound 2f Compound 2f Origin Cell Line IC₅₀ (μM)Origin Cell Line IC₅₀ (μM) Hematopoietic CCFR-CEM <0.006 Breast T-47D0.973 SR <0.007 MDA-MB-231 0.006 HL-60(TB) 0.017 MCF-7 3.75 RPMI-82260.017 Ovarian OVCAR-5 1.004 MOLT-4 0.161 OVCAR-8 0.068 Colon HCT-15 >10OVCAR-4 0.015 HCT-116 >10 IGROVI 0.01 KM12 1.37 SKOV-3 0.060 SW-620 3.31Prostate DU-145 0.232 HT29 0.452 PC-3 0.192 Lung NCI-H460 >10 MelanomaUACC-62 0.119 NCI-H322M 2.46 UACC-257 5.32 NCI-H23 0.013 LOX IMVI 0.004EKVX 0.014 M14 0.485 HOP-62 0.020 MALME-3M 0.132 Renal ACHN .0013 CNSSK-MEL-28 0.485 UO-31 <0.009 SNB-75 0.063 CAKI-1 <0.006 U251 0.259RXF-393 0.003 SF-268 0.69 SN12C 0.375 SNB-19 0.131 786-0 0.032 SF-5390.034 TK-10 0.078 Multi-drug NCI-ADR-RES 0.023 resistant

TABLE 5 Growth inhibitory activity of compound 2j in a panel of humantumor cell lines. Compound 2j Compound 2j Origin Cell Line IC₅₀ (μM)Origin Cell Line IC₅₀ (μM) Hematopoietic CCFR-CEM 0.01 Breast T-47D 0.12SR .008 MDA-MB-231 0.03 HL-60(TB) 0.04 MDA-MB-468 0.13 K562 1.25 BT-5490.76 RPMI-8226 0.08 Hs578T 0.22 MOLT-4 0.13 MCF-7 >3.0 Colon HCT-15 >3.0Ovarian OVCAR-5 0.49 HCT-116 >3.0 OVCAR-8 0.05 HCC-2998 2.26 OVCAR-40.05 KM12 0.63 OVCAR-3 0.13 SW-620 2.75 IGROV-1 0.02 COLO205 1.44SK-OV-3 0.32 HT29 0.65 Prostate DU-145 >3.0 Lung NCI-H522 0.016 PC-30.34 NCI-H460 <0.001 Melanoma UACC-62 0.29 NCI-H322M >3.0 UACC-257 >3.0NCI-H23 >3.0 LOX IMV1 <0.001 NCI-H226 1.41 M14 2.26 A549 >3.0MALME-3M >3.0 EKVX 0.02 MDA-MB-435 0.64 HOP-62 0.20 SK-MEL-2 0.92 HOP-920.02 SK-MEL-5 >3.0 Renal ACHN 0.01 SK-MEL-28 1.04 UO-31 0.005 CNS SNB-750.10 CAKI-1 0.007 U251 1.88 A498 0.096 SF-268 0.10 RXF-393 0.015 SNB-191.04 SN12C >3.0 SF-539 0.16 786-0 0.02 SF-295 0.07 TK-10 0.20 Multi-drugNCI-ADR/RES 0.02 resistant

Conclusions

Novel derivatives of the general formulae (1) and (2)-[(2-halophenyl),2,4-dichlorophenyl, 3,4-dichlorophenyl, 2,6-duchlorophenyl or2,5-dimethoxyphenyl]-2-haloalkanone-3-alkyl carboxylate and theircarboxylic acid derivatives were synthesized. Their chemical structureswere elucidated by infrared, nuclear magnetic resonance, massspectrometry and elemental analyses. The compounds were initially testedfor their growth inhibitory activity using human breast and colon tumorcell lines. As summarized in Tables 1-3, compounds were identified thatshowed potent tumor cell activity with IC₅₀ values in the low nanomolarto the low micromolar range. Compounds 2f and 2j were among the mostpotent to inhibit tumor cell growth in the low nanomolar range. Testingcompounds 2f and 2j in a large panel of human tumor cell lines showedthat many were highly sensitive with IC₅₀ values less than 10 nM.Interestingly, compound 2f showed no cytotoxicity to normal humanmammary epithelial cells (HMEC) up to a concentration of 10 μM, whichmay offer the potential for a large safety and selectivity index.Additionally, the multi-drug resistant tumor cell line (NCI/ADR-RES) wasalso highly sensitive to both compounds, which indicates the potentialof this class of compounds to evade common mechanisms of drugresistance. The mechanism of action of these compounds appears toinvolve cGMP phosphodiesterase inhibition, although they possess uniqueproperties compared with conventional PDE5 inhibitors, which do not haveanticancer activity. The enabling feature of this class of compounds forthe prevention or treatment of cancer was demonstrated by experimentsshowing strong in vivo antitumor activity of compound 2j in mice.

Experimental Section

All starting materials were commercially available and used withoutfurther purification. All reactions were carried out with the use ofstandard techniques under an inert atmosphere (N₂). The analyticalthin-layer chromatography (TLC) was carried out on E. Merck 60-F254pre-coated silica gel plates and components were usually visualizedusing UV light, Flash column chromatography was performed on silica gel60 (E. Merck, 230-400 mesh). Melting points were determined on BuchiMelting Point apparatus and are uncorrected. Proton NMR (¹H NMR) andcarbon NMR (¹³C NMR) spectra were recorded at ambient temperature onVarian Mercury VX-300 MHz spectrometer using tetramethylsilane asinternal standard, and proton chemical shifts are expressed in ppm inthe indicated solvent. The following abbreviations are used formultiplicity of NMR signals: s) singlet, brs) broad singlet, d) doublet,t) triplet, q) quadruplet, dd) double doublet, m) multiplet. Theelemental analyses were performed by the Microanalytical Unit, Facultyof Science, Cairo University; and are within ±0.4% of the theoreticalvalue, unless stated otherwise)

The following non-limiting examples further illustrate the presentdisclosure:

General procedure for the synthesis of Methyl1-aryl-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate (1a-r)

The appropriate tryptophan methyl ester (3.42 g, 15.7 mmol) and 2-bromobenzaldehyde, 2,5-dimethoxybenzaldehyde, 2,4-dichlorobenzaldehyde,3,4-dichlorobenzaldehyde or 2,6-dichlorobenzaldehyde (17.25 mmol) weredissolved in CH₂Cl₂ (10 mL) and cooled to 0° C. in an ice bath. To thissolution was added drop-wise TFA (1 mL), and the mixture was stirred atroom temperature for 4 days. The reaction mixture was then basified withdilute NH₄OH solution and extracted with CH₂Cl₂ (3×10 mL). The organiclayer was washed with water, brine, dried over anhydrous Na₂SO₄,filtered, and evaporated under reduced pressure. The residue waspurified and the isomers were separated by column chromatography onsilica gel eluting with CH₂Cl₂, to give first the appropriate cis-isomerfollowed by the trans-one. For the 2,6-dichlorobenzaldehyde, only thecis isomer was obtained and no chromatographic separation was carriedout.

Example 1 (1R,3R) Methyl1-(2-bromophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate (1a)

White solid; 33%; mp 105-106° C.; R_(f)=0.38 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(DMSO-d₆): δ 10.46 (s, 1H, NH), 7.7-7.68 (d, 1H, Ar), 7.46-7.43 (d, 1H,Ar), 7.33-7.20 (m, 4H, Ar), 7.04-6.93 (m, 2H, Ar), 5.64 (s, 1H, CHPh),3.95-3.91 (m, 1H, CHCOOCH₃), 3.68 (s, 3H, OCH₃), 3.07-3.03 (dd, 1H,CH_(a)H_(b)), 2.89-2.75 (m, 1H, CH_(a)H_(b)); ¹³C-NMR (DMSO-d₆): δ172.88, 140.75, 136.36, 134.16, 132.69, 130.74, 128.02, 126.44, 124.02,120.94, 118.54, 117.70, 111.32, 107.65, 56.03 (C1), 51.89 (C3), 51.85,25.26; MS: m/z 386 (M⁺+2), m/z 384 (M⁺, 100%); Anal. (C₁₉H₁₇BrN₂O₂) C,H, N.

Example 2 (1S,3R) Methyl1-(2-bromophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate (1b)

White solid; 41%; mp 194-195° C.; R_(f)=0.15 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(DMSO-d₆): δ 10.73 (s, 1H, NH), 7.69-7.62 (m, 1H, Ar), 7.49-7.47 (d, 1H,Ar), 7.25-7.22 (m, 4H, Ar), 7.05-6.98 (m, 2H, Ar), 5.67 (s, 1H, CHPh),3.68-3.63 (m, 4H, CHCOOCH₃+OCH₃), 3.14-3.08 (m, 1H, CH_(a)H_(b)),2.90-2.87 (dd, 1H, CH_(a)H_(b)); ¹³C-NMR (DMSO-d₆): δ 173.53, 141.11,136.2, 132.93, 130.37, 129.53, 127.47, 126.35, 124.09, 121.12, 118.51,117.81, 111.19, 107.86, 53.69 (C1), 51.83 (C3), 51.36, 24.89; MS: m/z386 (M⁺+2), m/z 384 (M⁺, 100%); Anal. (C₁₉H₁₇BrN₂O₂) C, H, N.

Example 3 (1S,3S) Methyl1-(2-bromophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate (1c)

White solid; 40%; mp 104-105° C.; R_(f)=0.39 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(DMSO-d₆): δ 10.46 (s, 1H, NH), 7.70-7.67 (d, 1H, Ar), 7.46-7.43 (d, 1H,Ar), 7.33-7.20 (m, 5H, Ar+NH), 7.04-6.93 (m, 2H, Ar), 5.64 (s, 1H,CHPh), 3.93 (m, 1H, CHCOOCH₃), 3.68 (s, 3H, OCH₃), 3.07-3.03 (dd, 1H,CH_(a)H_(b)), 2.90-2.80 (m, 1H, CH_(a)H_(b)); ¹³C-NMR (DMSO-d₆): δ172.80, 136.36, 134.07, 132.64, 130.74, 129.83, 128.01, 126.43, 124.02,120.93, 118.52, 117.67, 115.92, 111.30, 107.63, 56.61 (C1), 51.85 (C3),25.20; MS: m/z 386 (M⁺+2), m/z 384 (M⁺, 100%); Anal. (C₁₉H₁₇BrN₂O₂) C,H, N.

Example 4 (1R,3S) Methyl1-(2-bromophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate (1d)

White solid; 28%; mp 193-195° C.; R_(f)=0.14 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(DMSO-d₆): δ 10.71 (s, 1H, NH), 7.69-7.67 (t, 1H, Ar), 7.48-7.46 (d, 1H,Ar), 7.24-7.21 (d, 3H, Ar), 7.07-6.95 (m, 2H, Ar), 6.76-6.5 (m, 1H, Ar),5.66 (s, 1H, CHPh), 3.67-3.62 (m, 4H, CHCOOCH₃+OCH₃), 3.13-3.07 (m, 1H,CH_(a)H_(b)), 2.89-2.80 (dd, 1H, CH_(a)H_(b)); ¹³C-NMR (DMSO-d₆): δ173.51, 141.10, 136.19, 132.91, 130.35, 129.50, 127.45, 126.34, 124.06,121.09, 118.48, 117.77, 111.16, 107.83, 53.64 (C1), 51.79 (C3), 51.35,24.89; m/z 386 (M⁺+2), m/z 384 (M⁺, 100%); Anal. (C₁₉H₁₇BrN₂O₂) C, H, N.

Example 5 (1R,3R) Methyl1-(2,5-dimethoxyphenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(Ie)

Buff solid; 25%; mp 102-105° C.; R_(f): 0.39 (CH₂Cl₂/MeOH 98:2); ¹H-NMR(DMSO-d₆): δ 7.79 (brs, 1H, NH), 7.53-7.50 (d, 1H, Ar), 7.25-6.82 (m,6H, Ar), 5.71 (s, 1H, CHPh), 4.02-3.97 (m, 1H, CHCOOCH₃), 3.87 (s, 3H,OCH₃), 3.83 (s, 3H, OCH₃), 3.71 (s, 3H, OCH₃), 3.37-3.17 (m, 1H,CHaH_(b)), 3.05-3.00 (m, 1H, CH_(a)Hb). ¹³C-NMR (DMSO-d₆): δ 173.1,154.1, 151.3, 135.9, 134.5, 127.1, 121.6, 119.4, 117.9, 114.7, 114.1,112.2, 110.9, 108.2, 56.9, 56.4, 55.8 (C1), 52.2 (C3), 52.0, 25.4; MS:m/z 366 (M⁺, 100); Anal (C₂₁H₂₂N₂O₄) C, H, N.

Example 6 (1S,3R) Methyl1-(2,5-dimethoxyphenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(If)

Yellow solid; 45%; mp 139-142° C.; R_(f): 0.18 (CH₂Cl₂/MeOH 98:2);¹H-NMR (DMSO-d₆): δ 7.93 (s, 1H, NH), 7.56-7.53 (d, 1H, Ar), 7.25-6.62(m, 6H, Ar), 5.81 (s, 1H, CHPh), 3.97-3.95 (m, 1H, CHCOOCH₃), 3.93 (s,3H, OCH₃), 3.75 (s, 3H, OCH₃), 3.66 (s, 3H, OCH₃), 3.28-3.21 (dd, 1H,CHaH_(b)), 3.10-3.02 (m, 1H, CH_(a)Hb), ¹³C-NMR (DMSO-d₆): δ 173.8,153.5, 151, 136.3, 132.4, 127.8, 126.9, 125.8, 121.8, 119.2, 118, 115.8,112.6, 111.5, 108.5, 56.0, 55.7, 52.4, 52.2 (C1), 49.4 (C3), 29.7; MS:m/z 366 (M⁺, 100); Anal. (C₂₁H₂₂N₂O₄) C, H, N.

Example 7 (1S,3S) Methyl1-(2,5-dimethoxyphenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(Ig)

Yellow solid; 25%; mp 100-102° C.; R_(f) 0.38 (CH₂Cl₂/MeOH 98:2); ¹H-NMR(DMSO-d₆): δ 7.78 (s, 1H, NH), 7.53-7.51 (d, 1H, Ar), 7.27-6.82 (m, 6H,Ar), 5.69 (s, 1H, CHPh), 4.02-3.97 (dd, 1H, CHCOOCH₃), 3.88 (s, 3H,OCH₃), 3.83 (s, 3H, OCH₃), 3.71 (s, 3H, OCH₃), 3.25-3.18 (m, 1H,CHaH_(b)), 2.95-3.05 (m, 1H, CH_(a)Hb); ¹³C-NMR (DMSO-d₆): δ 173.1,154.2, 151.3, 135.9, 134.4, 127, 121.6, 119.4, 117.9, 114.7, 114.1,112.3, 110.8, 108.1, 56.9, 56.4, 55.8 (C1), 52.2 (C3), 52, 25.5; MS: m/z366 (M⁺, 100); Anal. (C₂₁H₂₂N₂O₄) C, H, N.

Example 8 (1R,3S) Methyl1-(2,5-dimethoxyphenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(Ih)

Yellow solid; 45%; mp 138-141° C.; R_(f.) 0.17 (CH₂Cl₂/MeOH 98:2);¹H-NMR (DMSO-d₆): δ 7.96 (brs, 1H, NH), 7.56-7.53 (d, 1H, Ar), 7.18-6.61(m, 6H, Ar), 5.82 (s, 1H, CHPh), 3.98-3.96 (m, 1H, CHCOOCH₃), 3.920 (s,3H, OCH₃), 3.75 (s, 3H, OCH₃), 3.66 (s, 3H, OCH₃), 3.29-3.22 (m, 1H,CHaH_(b)), 3.04-3.12 (m, 1H, CH_(a)Hb), ¹³C-NMR (DMSO-d₆): δ 173.9,153.5, 151.1, 136.1, 132.8, 131.5, 126.9, 121.7, 119.2, 118, 115.6,112.5, 111.6, 110.9, 108.7, 56.1, 55.7, 52.5, 52.0 (C1), 49.1 (C3),24.9; MS: m/z 366 (M⁺, 100); Anal. (C₂₁H₂₂N₂O₄) C, H, N.

Example 9 (1R,3R)Methyl-1-(2,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(1i)

Yellow powder; 35%; m.p: 88-92° C.; R_(f)=0.19 (CH₂Cl₂); IR (cm⁻¹): 3406(—NH—), 1734(—CO—); ¹H-NMR: δ 7.48 (s, 1H, NH), 7.51 (d, 1H, J=7.0 Hz,Ar), 7.35 (d, 1H, J=7.0 Hz, Ar), 7.26-7.15 (m, 4H, Ar+NH), 7.04-6.93 (m,2H, Ar), 5.82 (s, 1H, CHPh), 4.03-4.00 (m, 1H, CHCOOCH₃), 3.83 (s, 3H,OCH₃), 3.25 (dd, 1H, J=4.3/7.0 Hz, CH_(a)H_(b)), 3.23-3.04 (m, 1H,CH_(a)H_(b)); ¹³C-NMR: δ 172.82, 136.16, 135.01, 134.11, 132.10, 131.23,130.02, 128.06, 126.81, 123.40, 122.26, 119.83, 118.28, 110.97, 109.33,56.55 (C1), 54.53, 52.40 (C3), 25.28; MS: m/z 378 (M⁺+4), m/z 376(M⁺+2), m/z 374 (M⁺), m/z 217 (100%); Anal. (C₁₉H₁₆Cl₂N₂O₂), C, H, N.

Example 10 (1S,3R) Methyl1-(2,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(1j)

Yellow powder; 34%; m.p: 130-133° C.; R_(f)=0.12 (CH₂Cl₂); IR (cm⁻¹):3388 (—NH—), 1735(—CO—); ¹H-NMR: δ 7.64 (s, 1H, NH), 7.56 (d, 1H, J=7.0Hz, Ar), 7.47 (s, 1H, NH), 7.00 (d, 1H, J=7.0 Hz, Ar), 7.27-7.12 (m, 5H,Ar), 5.97 (s, 1H, CHPh), 3.92-3.88 (m, 1H, CHCOOCH₃), 3.74 (s, 3H,OCH₃), 3.30 (dd, 1H, J=4.6/7.6 Hz, CH_(a)H_(b)), 3.17-3.15 (m, 1H,CH_(a)H_(b)); ¹³C-NMR: δ 173.09, 137.15, 136.29, 134.73, 134.51, 131.16,130.83, 129.78, 127.27, 126.63, 122.48, 119.82, 118.38, 111.08, 109.43,52.40 (C1), 52.27, 51.15 (C3), 24.39; MS: m/z 378 (M⁺+4), m/z 376(M⁺+2), m/z 374 (M⁺, 100%); Anal. (C₁₉H₁₆Cl₂N₂O₂), C, H, N.

Example 11 (1S,3S)Methyl1-(2,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(1k)

White powder; 45%; m.p: 90-92° C.; R_(f)=0.19 (CH₂Cl₂); IR (cm⁻¹): 3433(—NH—), 1728 (—CO—); ¹H NMR: δ 7.90 (s, 1H, NH), 7.54-7.43 (m, 2H, Ar),7.38 (d, 1H, J=7.2 Hz, Ar), 7.26-7.10 (m, 5H, Ar+NH), 5.8 (s, 1H, CHPh),3.90-3.87 (m, 1H, CHCOOCH₃), 3.82 (s, 3H, OCH₃), 3.25 (dd, 1H, J=4.3/7.6Hz, CHaH_(b)), 3.01-2.90 (m, 1H, CH_(a)Hb); ¹³C-NMR: δ 172.90, 136.15,135.10, 134.65, 133.15, 132.12, 131.45, 130.0, 129.38, 128.04, 126.85,122.01, 119.8, 115.92, 110.96, 109.37, 56.57 (C1), 51.36 (C3), 25.38;MS: m/z 378 (M⁺+4), m/z 376 (M⁺+2), m/z 374 (M+, 100%); Anal.(C₁₉H₁₆Cl₂N₂O₂), C, H, N.

Example 12 (1R,3S)Methyl1-(2,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(1l)

White powder; 30%; m.p: 128-130° C.; R_(f)=0.12 (CH₂Cl₂); IR (cm⁻¹):3396 (—NH—), 1739 (—CO—); ¹H NMR: δ 7.80 (s, 1H, NH), 7.49 (s, 1H, NH),7.46 (d, 1H, J=7.2 Hz, Ar), 7.19-7.11 (m, 5H, Ar), 6.94 (d, 1H, J=7.0Hz, Ar), 5.30 (s, 1H, CHPh), 3.74 (s, 3H, OCH₃), 3.68-3.62 (m, 1H,CHCOOCH₃), 3.30 (dd, 1H, J=4.6/7.6 Hz, CHaH_(b)), 3.16-3.14 (m, 1H,CH_(a)Hb); ¹³C-NMR: δ 173.17, 136.27, 134.70, 134.50, 131.12, 131.0,129.77, 127.27, 126.65, 122.46, 120.01, 119.82, 118.39, 111.06, 109.46,52.38 (C1), 51.28 (C3), 51.14; MS: m/z 378 (M⁺+4), m/z 376 (M⁺+2), m/z374 (M+, 100%); Anal. (C₁₉H₁₆Cl₂N₂O₂), C, H, N.

Example 13 (1R,3R)Methyl-1-(3,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(1m)

Yellow powder; 35%; mp: 90-92° C.; R_(f)=0.15 (CH₂Cl₂); IR (cm⁻¹): 3396(—NH—), 1734 (—CO—); ¹H NMR: δ 9.0 (s, 1H, NH), 7.51-7.44 (m, 5H,Ar+NH), 7.25 (d, 1H, J=7.2 Hz, Ar), 7.20-7.10 (m, 2H, Ar), 5.23 (s, 1H,CHPh), 3.96 (dd, J=4.0/7.6 Hz, 1H, CH_(a)H_(b,)), 3.82 (s, 3H, OCH₃),3.05-3.01 (m, 1H, CHCOOCH₃), 3.00-2.95 (m, 1H, CH_(a)H_(b)); ¹³C-NMR: δ172.88, 140.98, 136.25, 133.30, 133.12, 132.74, 130.92, 130.58, 127.94,126.90, 122.35, 119.89, 118.37, 111.03, 109.34, 57.73 (C1), 56.64, 52.42(C3), 25.43; MS: m/z 378 (M⁺+4), m/z 376 (M⁺+2), m/z 374 (M⁺, 100%);Anal.; [α]²⁰ _(D)=+58.35 (c=0.1, EtOH).

Example 14 (1S,3R)Methyl-1-(3,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(1n)

Yellow powder; 20%; m.p: 140-143° C.; R_(f)=0.10 (CH₂Cl₂); IR (cm⁻¹):3345 (—NH—), 1710 (—CO—); ¹HNMR: δ 7.55 (d, 1H, J=7.2 Hz, Ar), 7.50-7.02(m, 3H, Ar), 7.20-7.10 (m, 5H, Ar), 5.49 (s, 1H, CHPh), 3.98-3.95 (m,1H, CHCOOCH₃), 3.73 (s, 3H, OCH₃), 3.37 (d, 1H, J=8.0 Hz, CH_(a)H_(b)),3.29 (dd, 1H, J=4.6/8.0 Hz, CH_(a)H_(b,)); ¹³C-NMR: δ 172.87, 148.02,136.27, 135.02, 132.95, 132.55, 130.72, 130.57, 128.02, 126.69, 122.51,119.85, 118.45, 111.09, 108.57, 53.94 (C1), 52.59 (C3), 52.40, 24.17;MS: m/z 378 (M⁺+4), m/z 376 (M⁺+2), m/z 374 (M⁺, 100%); Anal.; [α]²⁰_(D)=+ 69.5 (c=0.1, EtOH).

Example 15 (1S,3S)Methyl-1-(3,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(1o)

Yellow powder; 38%; m.p: 88-91° C.; R_(f)=0.15 (CH₂Cl₂); IR (cm⁻¹): 3395(—NH—), 1734 (—CO—); ¹H NMR: δ 7.51-7.44 (m, 51-1, Ar+NH), 7.25 (d, 1H,J=7.2 Hz, Ar), 7.20-7.10 (m, 2H, Ar), 5.24 (s, 1H, CHPh), 3.95 (dd, 1H,J=4.0/8.0 Hz, CHCOOCH₃), (s, 3H, OCH₃), 3.25 (dd, 1H, J=4.6/7.6 Hz,CH_(a)H_(b,)) 3.05-2.95 (m, 1H, CH_(a)H_(b)); ¹³C-NMR: δ 172.78, 140.81,136.27, 133.16, 133.12, 132.78, 130.919, 130.62, 127.99, 127.51, 126.88,122.37, 119.90, 118.37, 111.04, 109.32, 57.70 (C1), 52.42 (C3), 25.36;MS: m/z 378 (M⁺+4), m/z 376 (M⁺+2), m/z 374 (M⁺, 100%); Anal.(C₁₉H₁₆Cl₂N₂O₂), C, H, N; [α]²⁰ _(D)=58.35 (c=0.1, EtOH).

Example 16(1R,3S)Methyl-1-(3,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(1p)

Yellow powder; 25%; m.p: 139-142° C.; R_(f)=0.10 (CH₂Cl₂); IR (cm⁻¹):3386 (—NH—), 1731 (—CO—); ¹H NMR: δ 7.55 (d, 2H, J=7.0 Hz, Ar+NH),7.40-7.35 (m, 3H, Ar+NH), 7.27 (d, 2H, J=7.0 Hz, Ar), 7.21-7.10 (m, 2H,Ar), 5.45 (s, 1H, CHPh), 3.97-3.93 (m, 1H, CHCOOCH₃), 3.73 (s, 3H,OCH₃), 3.31-2.26 (m, 1H, CH_(a)H_(b)), 3.18 (dd, 1H, J=4.6/8.0 Hz,CH_(a)H_(b,)); ¹³C-NMR: δ 173.13, 139.96, 136.04, 135.80, 132.57,130.45, 130.22, 129.07, 128.84, 126.98, 124.06, 121.91, 119.77, 118.01,110.99, 108.63, 53.20 (C1), 52.36 (C3), 25.72; MS: m/z 378 (M⁺+4), m/z376 (M⁺+2), m/z 374 (M⁺, 100%); Anal. (C₁₉H₁₆Cl₂N₂O₂), C, H, N; [α]²⁰_(D)=−69.5 (c=0.1, EtOH).

Example 17 (1R,3R) Methyl1-(2,6-dichlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3 carboxylate(1q)

Red powder; 55%; m.p: 102-105° C.; R_(f)=0.13 (100% CH₂Cl₂); IR (cm⁻¹):3389 (—NH—), 1729 (—CO—); ¹H-NMR: δ 7.58-7.52 (m, 3H, Ar+NH), 7.28-7.27(m, 3H, Ar+NH), 7.26-7.25 (m, 3H, Ar), 6.29 (s, 1H, CHPh), 4.14-4.06 (m,1H, CHCOOCH₃), 3.85 (s, 3H, OCH₃), 3.32 (dd, 1H, J=4.0/8.0 Hz,CH_(a)H_(b)), 3.03-2.99 (m, 1H, CH_(a)H_(b)); ¹³C-NMR: δ 172.25, 138.07,136.20, 135.88, 134.37, 130.47, 129.40, 127.84, 126.60, 121.82, 119.66,118.07, 112.06, 111.02, 56.83 (C1), 54.07, 52.21 (C3), 37.04, 27.15; MS:m/z 378 (M⁺+4), m/z 376 (M⁺+2), m/z 374 (M⁺), m/z 217 (100%); Anal.(C₁₉H₁₆Cl₂N₂O₂), C, H, N.

Example 18 (1S,3S) Methyl1-(2,6-dichlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(1r)

Red powder; 57%; m.p: 101-103° C.; R_(f)=0.13 (100% CH₂Cl₂); IR (cm⁻¹):3386 (—NH—), 1730 (—CO—); ¹H-NMR: δ 7.51-7.50 (m, 3H, Ar+NH), 7.43 (d,1H, J=7.2 Hz, Ar), 7.26-7.25 (m, 31-1, Ar+NH), 7.13-7.11 (m, 2H, Ar),6.21 (s, 1H, CHPh), 4.07-4.04 (m, 1H, CHCOOCH₃), 3.83 (s, 3H, OCH₃),3.25 (dd, 1H, J=4.0/8.0 Hz, CH_(a)H_(b)), 2.99-2.96 (m, 1H,CH_(a)H_(b)); ¹³C-NMR: δ 173.13, 136.96, 135.80, 134.3, 132.57, 130.46,129.08, 128.84, 126.98, 121.91, 119.77, 118.01, 110.99, 108.64, 57.21(C1), 54.21, 52.36 (C3), 36.88, 25.72; MS: m/z 378 (M⁺+4), m/z 376(M⁺+2), m/z 374 (M⁺, 100%); Anal. (C₁₉H₁₆Cl₂N₂O₂), C, H, N.

General procedure for Methyl1-aryl-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2a-r)

To a stirred solution of the appropriate β-carboline 1a-r (5.7 mmol) andNaHCO₃ (6.89 mmol) in CHCl₃ (40 mL) chloroacetyl chloride (1.09 mL,13.69 mmol) was added dropwise under ice cooling. The mixture was thenstirred at room temperature for 2 hours. The mixture was diluted withCH₂Cl₂, and washed with a solution of NaHCO₃. The organic layer wasdried over Na₂SO₄ and evaporated under reduced pressure. The residue wassubjected to column chromatography using CH₂Cl₂ as an eluent, thenrecrystallized from ethanol.

Example 19 Methyl(1R,3R)-1-(2-bromophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2a)

White solid; 88%; mp 113-115° C.; R_(f)=0.68 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(CDCl₃): δ 8.00 (br s, 1H, NH), 7.88-7.85 (d, 1H, Ar), 7.57-7.54 (d, 1H,Ar), 7.31-7.14 (m, 6H, Ar), 6.73 (s, 1H, CHPh), 5.18-5.14 (m, 1H,CHCOOCH₃), 4.19-4.15 (m, 2H, COCH₂Cl), 3.64 (br s, 3H, OCH₃), 3.37-3.28(m, 2H, CH₂); MS: m/z 462 (M⁺+2), m/z 460 (M⁺), m/z 385 (100%); Anal.(C₂₁H₁₈BrClN₂O₃). C, H, N

Example 20 Methyl(1S,3R)-1-(2-bromophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2b)

White solid; 74%; mp 132-135° C.; R_(f)=0.58 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(CDCl₃): δ 8.22 (s, 1H, NH), 7.61-7.50 (m, 2H, Ar), 7.28-7.08 (m, 6H,Ar), 6.65 (s, 1H, CHPh), 5.31 (br s, 1H, CHCOOCH₃), 4.19-4.06 (m, 2H,COCH₂Cl), 3.82-3.72 (m, 5H, OCH₃+CH₂); MS: m/z 462 (M⁺+2), m/z 460 (M⁺),m/z 383 (100%); Anal. (C₂₁H₁₈BrClN₂O₃). C, H, N.

Example 21 Methyl(1S,3S)-1-(2-bromophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2c)

White solid; 83%; mp 112-113° C.; R_(f)=0.68 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(CDCl₃): δ 7.97 (br s, 1H, NH), 7.68-7.65 (d, 1H, Ar), 7.57-7.54 (d, 1H,Ar), 7.31-7.12 (m, 6H, Ar), 6.73 (s, 1H, CHPh), 5.18-5.14 (m, 1H,CHCOOCH₃), 4.19-4.15 (m, 2H, COCH₂Cl), 3.64 (br s, 3H, OCH₃), 3.37-3.26(m, 21-1, CH₂); MS: m/z 462 (M⁺+2), m/z 460 (M⁺), m/z 383 (100%); Anal.(C₂₁H₁₈BrClN₂O₃). C, H, N.

Example 22 Methyl(1R,3S)-1-(2-bromophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2d)

White solid; 81%; mp 132-133° C.; R_(f)=0.60 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(CDCl₃): δ 8.22 (s, 1H, NH), 7.62-7.50 (m, 2H, Ar), 7.29-7.08 (m, 6H,Ar), 6.65 (s, 1H, CHPh), 5.31 (br s, 1H, CHCOOCH₃), 4.19-4.06 (m, 2H,COCH₂Cl), 3.74-3.65 (m, 5H, OCH₃+CH₂); MS: m/z 462 (M⁺+2), m/z 460 (M⁺),m/z 385 (100%); Anal. (C₂₁H₁₈BrClN₂O₃). C, H, N.

Example 23 Methyl(1R,3R)-1-(2,5-dimethoxyphenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2e)

White solid; 85%; mp 108-111° C.; R_(f)=0.66 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(CDCl₃): δ8.08 (br s, 1H, NH), 7.56-7.54 (d, 1H, Ar), 7.31-7.28 (m, 2H,Ar), 7.27-7.13 (m, 2H, Ar), 6.93-6.91 (d, 1H, Ar), 6.82-6.80 (m, 2H,Ar), 6.79 (brs, 1H, CHPh), 5.33 (brs, 1H, CHCOOCH₃), 4.28 (brs, 21-1,COCH₂Cl), 3.98 (s, 3H, OCH₃), 3.69 (s, 3H, OCH₃), 3.50 (s, 3H, OCH₃),3.44-3.19 (m, 2H, CH₂); MS: m/z 442 (M⁺), m/z 365 (100); Anal.(C₂₃H₂₃ClN₂O₅). C, H, N.

Example 24 Methyl(1S,3R)-1-(2,5-dimethoxyphenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2f)

White solid; 89%; mp 232-232° C.; R_(f)=0.49 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(CDCl₃): δ8.26 (br s, 1H, NH), 7.51-7.48 (d, 1H, Ar), 7.27-7.24 (m, 2H,Ar), 7.17-7.12 (m, 2H, Ar), 6.96 (brs, 1H, Ar), 6.81-6.79 (m, 1H, Ar),6.60 (brs, 1H, CHPh), 5.52 (brs, 1H, CHCOOCH₃), 4.23 (s, 2H, COCH₂Cl),4.05 (s, 3H, OCH₃), 3.69 (s, 3H, OCH₃), 164 (s, 3H, OCH₃), 3.42-3.10 (m,2H, CH₂); MS: m/z 442 (M⁺), m/z 365 (100); Anal (C₂₃H₂₃ClN₂O₅). C, H, N.

Example 25 Methyl(1S,3S)-1-(2,5-dimethoxyphenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2g)

White solid; 83%; mp 105-108° C.; R_(f)=0.65 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(CDCl₃): δ 8.05 (br s, 1H, NH), 7.56-7.54 (d, 1H, Ar), 7.27-7.14 (m, 4H,Ar), 6.94-6.91 (d, 1H, Ar), 6.82-6.80 (m, 1H, Ar), 6.58 (brs, 1H, CHPh),5.38 (brs, 1H, CHCOOCH₃), 4.52 (brs, 2H, COCH₂Cl), 4.05 (s, 3H, OCH₃),3.66 (s, 3H, OCH₃), 3.51 (s, 3H, OCH₃), 3.45-3.25 (m, 2H, CH₂); MS: m/z442 (M⁺), m/z 365 (100); Anal (C₂₃H₂₃ClN₂O₅). C, H, N.

Example 26 Methyl(1R,3S)-1-(2,5-dimethoxyphenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-4H-β-carboline-3-carboxylate(2h)

White solid; 88%; mp 228-230° C.; R_(f)=0.49 (CH₂Cl₂/MeOH 99:1); ¹H-NMR(CDCl₃): δ 8.24 (br s, 1H, NH), 7.51-7.48 (d, 1H, Ar), 7.27-1-7.25 (m,2H, Ar), 7.23-7.12 (m, 2H, Ar), 6.93-6.91 (d, 1H, Ar), 6.82-6.80 (m, 1H,Ar), 6.61 (brs, 1H, 1H, CHPh), 5.53 (brs, 1H, CHCOOCH₃), 4.23 (brs, 2H,COCH₂Cl), 4.06 (s, 3H, OCH₃), 3.69 (s, 3H, OCH₃), 3.64 (s, 3H, OCH₃),3.40-3.19 (m, 2H, CH₂); MS: m/z 442 (M⁺), m/z 365 (100); Anal.(C₂₃H₂₃ClN₂O₅). C, H, N.

Example 27 (1R,3R) Methyl1-(2,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2i)

Orange powder; 78%; m.p: 215-218° C.; R_(f)=0.53 (CH₂Cl₂); IR (cm⁻¹):3294 (—NH—), 1731, 1666 (—CO—); ¹H-NMR: δ 7.50 (s, 1H, NH), 7.30-7.16(m, 7H, Ar), 6.63 (s, 1H, CHPh), 4.53 (s, 1H, CHCOOCH₃), 4.20-4.15 (m,2H, COCH₂Cl), 3.64 (s, 3H, OCH₃), 3.43-3.39 (m, 2H, CH₂); MS: m/z 454(M⁺+4), m/z 452 (M⁺+2), m/z 450 (M⁺), m/z 373 (100%); Anal.(C₂₁H₁₇Cl₃N₂O₃), C, H, N.

Example 28 (1S,3R) Methyl1-(2,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2j)

Green powder; 80%; m.p: 106-109° C.; R_(f)=0.37 (100% CH₂Cl₂); IR(cm⁻¹): 3370 (—NH—), 1739, 1668 (—CO—); ¹H-NMR: δ 8.08 (s, 1H, NH), 7.51(d, 1H, J=7.2, Ar), 7.28-7.11 (m, 6H, Ar), 6.57 (s, 1H, CHPh), 5.30 (brs, 1H, CHCOOCH₃), 4.18-4.10 (m, 2H, COCH₂Cl), 3.64 (s, 3H, OCH₃),3.53-3.49 (m, 2H, CH₂); MS: m/z 454 (M⁺+4), m/z 452 (M⁺+2), m/z 450(M⁺), m/z 373 (100%); Anal. (C₂₁H₁₇Cl₃N₂O₃), C, H, N.

Example 29 (1S,3S) Methyl1-(2,4dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2k)

White powder; 88%; m.p: 215-218° C.; R_(f)=0.52 (CH₂Cl₂); IR (cm⁻¹):3315 (—NH—), 1737 (—CO—), 1664 (—CO—); ¹H NMR: δ 7.50 (brs, 1H, NH),7.26-7.15 (m, 7H, Ar), 6.57 (s, 1H, CHPh), 4.52 (s, 1H, CHCOOCH₃),4.20-4.15 (m, 2H, COCH₂Cl), 3.63 (br s, 3H, OCH₃), 3.49-3.48 (m, 2H,CH₂); MS: m/z 454 (M⁺+4), m/z 452 (M⁺+2), m/z 450 (M⁺, 100%); Anal.(C₂₁H₁₇Cl₃N₂O₃), C, H, N.

Example 30 (1R,3S) Methyl1-(2,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carsboline-3-carboxylate(2l)

White powder; 75%; m.p: 109-112° C.; R_(f)=0.37 (CH₂Cl₂); IR (cm⁻¹):3347 (—NH—), 1739 (—CO—), 1668 (—CO—); ¹H NMR: δ 7.80 (brs, 1H, NH),7.39 (d, 1H, J=8.0 Hz, Ar), 7.18-7.12 (m, 6H, Ar), 6:54 (s, 1H, CHPh),5.56 (d, 1H, J=7.0 Hz, CHCOOCH₃), 3.95-393 (m, 2H, COCH₂Cl), 3.72 (s,3H, OCH₃), 3.31 (dd, 1H, J=3.6/7.6 Hz, CH_(a)CH_(b)), 3.18 (dd, 1H,J=3.6/7.6 Hz, CH_(a)CH_(b)); MS: m/z 454 (M⁺+4), m/z 452 (M⁺+2), m/z 450(M⁺), m/z 373 (100%); Anal. (C₂₁H₁₇C₁₃N₂O₃), C, H, N.

Example 31 (1R,3R) Methyl1-(3,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2m)

White powder; 86%; m.p: 283-285° C.; R_(f)=0.47 (CH₂Cl₂); IR (cm⁻¹):3283 (—NH—), 1737 (—CO—), 1662 (—CO—); ¹H NMR: δ 8.9 (s, 1H, NH), 7.60(d, 2H, J=7.6 Hz, Ar), 7.32-7.16 (m, 5H, Ar), 6.83 (s, 1H, CHPh), 4.99(d, 1H, J=7.0 Hz, CHCOOCH₃), 4.35 (m, COCH₂Cl), 4.22 (dd, 1H, J=3.6/7.6Hz, CH_(a)CH_(b)), 3.70 (dd, 1H, J=3.6/7.6 Hz, CH_(a)CH_(b)), 3.49 (s,3H, OCH₃); MS: m/z 454 (M⁺+4), m/z 452 (M⁺+2), m/z 450 (M⁺, 100%); Anal.(C₂₁H₁₇Cl₃N₂O₃), C, H, N.

Example 32 (1S,3R) Methyl1-(3,4dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2n)

White powder; 79%; m.p: 163-165° C.; R_(f)=0.22 (CH₂Cl₂); IR (cm⁻¹):3387 (—NH—), 1734 (—CO—), 1663 (—CO—); ¹H NMR: δ 8.9 (s, 1H, NH), 7.77(d, 2H, J=8.2 Hz, Ar), 7.62-7.42 (m, 2H, Ar), 7.26-7.23 (m, 3H, Ar),6.72 (s, 1H, CHPh), 4.99-4.97 (m, 1H, CHCOOCH₃), 4.36-4.34 (m, 2H,COCH₂Cl), 4.10 (s, 1H, CH_(a)CH_(b)), 3.75 (dd, 1H, J=3.6/7.6 Hz,CH_(a)CH_(b)), 3.49 (s, 3H, OCH₃); MS an/z 454 (M⁺+4), m/z 452 (M⁺+2),m/z 450 (M⁺, 100%); Anal. (C₂₁H₁₇Cl₃N₂O₃), C, H, N.

Example 33 (1S,3S) Methyl1-(3,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate(2o)

White powder; 84%; m.p: 283-284° C.; R_(f)=0.45 (CH₂Cl₂); IR (cm⁻¹):3281 (—NH—), 1736 (—CO—), 1662 (—CO—); ¹H NMR: δ 8.9 (s, 1H, NH), 7.77(d, 2H, J=8.0 Hz, Ar), 7.61 (d, 2H, J=8.0 Hz, Ar), 7.32-7.17 (m, 3H,Ar), 6.83 (s, 1H, CHPh), 4.98 (d, 1H, J=7.0 Hz, CHCOOCH₃), 4.36-4.34 (m,2H, COCH₂Cl), 4.21 (d, 1H, J=3.4/7.6 Hz, CH_(a)CH_(b)), 3.70 (dd, 1H,J=3.4/7.6 Hz, CH_(a)CH_(b)), 3.46 (s, 3H, OCH₃); MS: m/z 454 (M⁺+4), m/z452 (M⁺+2), m/z 450 (M⁺, 100%); Anal. (C₂₁H₁₇Cl₃N₂O₃), C, H, N.

Example 34 (1R,3S) Methyl 1-(3,4-dichloro-phenyl)-2(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate (2p)

White powder; 77%; m.p: 162-165° C.; R_(f)=0.24 (CH₂Cl₂); IR (cm⁻¹):3275 (—NH—), 1738, 1663 (—CO—); ¹H NMR: δ 7.9 (s, 1H, NH), 7.75 (d, 2H,J=7.6 Hz, Ar), 7.22-7.12 (m, 5H, Ar), 6.03 (s, 1H, CHPh), 4.16 (d, 1H,J=7.0 Hz, CHCOOCH₃), 4.13-4.06 (m, 2H, COCH₂Cl), 3.61-3.58 (m, 2H, CH₂),3.47 (s, 3H, OCH₃); MS: m/z 454 (M⁺+4), m/z 452 (M⁺+2), m/z 450 (M⁺,100%); Anal. (C₂₁H₁₇Cl₃N₂O₃), C, H, N.

Example 35 (1R,3R) Methyl1-(2,6-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro1H-β-carboline-3-carboxylate (2q)

Brown powder; 76%; m.p: 147-150° C.; R_(f)=0.44 (100% CH₂Cl₂); IR(cm⁻¹): 3304 (—NH—), 1739, 1666 (—CO—); ¹H-NMR: δ 8.98 (s, 1H, NH),7.75-7.15 (m, 7H, Ar), 6.90 (s, 1H, CHPh), 4.99 (s 1H, CHCOOCH₃),4.25-4.22 (m, 2H, COCH₂Cl), 3.67 (s, 3H, OCH₃), 3.37-3.16 (m, 2H, CH₂);MS: m/z 454 (M⁺+4), m/z 452 (M⁺+2), m/z 450 (M⁺), m/z 375 (100%); Anal.(C₂₁H₁₇Cl₃N₂O₃), C, H, N.

Example 36 (1S,3S) Methyl1-(2,6-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxyl(2r)

Brown powder; 78%; m.p: 153-156° C.; R_(f)=0.42 (100% CH₂Cl₂); IR(cm⁻¹): 3309 (—NH—), 1737, 1666 (—CO—); ¹H-NMR: δ 8.98 (s, 1H, NH),7.30-7.15 (m, 7H, Ar), 6.90 (s, 1H, CHPh), 4.99 (s, 1H, CHCOOCH₃),4.25-4.21 (m, 2H, COCH₂Cl), 3.68 (s, 3H, OCH₃), 3.22-3.17 (m, 2H, CH₂);MS: m/z 454 (M⁺+4), m/z 452 (M⁺+2), m/z 450 (M⁺), m/z 375 (100%); Anal.(C₂₁H₁₇Cl₃N₂O₃), C, H, N.

Cell Cultures

All human tumor cell lines were obtained from ATCC and grown understandard cell culture conditions at 37° C. in a humidified atmospherewith 5% CO₂. Cells were grown in RPMI 1640 supplemented with 5% fetalbovine serum 2 mM glutamine, 100 units/ml penicillin, 100 units/mlstreptomycin, and 0.25 μg/ml amphotericin. Cells were harvested at 70 to90% confluence with trypsin/EDTA and used immediately Cell count andviability was determined by Trypan blue staining followed byhemocytometry. Only cultures displaying >95% cell viability were usedfor experiments. HMEC were grown according to the specifications fromthe supplier (Lonzo).

Growth Assays

Tissue culture microtiter 96-well plates were seeded with cells at adensity of 5,000 cells/well. The plates were incubated for 18-24 hoursprior to any treatment. All test compounds were solubilized in 100% DMSOand diluted with media to obtain a final DMSO concentration of 0.1%.Cell viability was measured 72 hours after treatment by the Cell TiterGlo Assay (Promega), which is a luminescent assay that is an indicatorof live cells as a function of metabolic activity and ATP content. Theassay was performed according to manufacturer's specifications.Luminescence was measured by a Perkin Elmer Victor® multi-label platereader.⁶

cGMP Assay

Cells were seeded at a density of 1×10⁶ cells per 10 cm tissue culturedish, incubated for 48 hours, and treated with the specified compound orvehicle control. After 30 minutes of treatment, cells were lysed andassayed for cGMP content using the cGMP Direct Biotrak EIA kit (GEBiosciences). The assay was performed according to the manufacturer'sspecifications. Optical density was measured at 630 nm using a Synergy4(BioTek) plate reader.

Experimental Design and Data Analysis

The potency of compounds to inhibit tumor cell growth was expressed byan IC₅₀ value (50% inhibitory concentration). The IC₅₀ value wasdetermined by testing a range of 8 concentrations with at least fourreplicates per concentration. Dose response curves were analyzed usingPrism™ 4 software (GraphPad) to calculate IC₅₀ values using a fourparameter logistic equation.

Formulations

Compounds of the present disclosure can be administered by anyconventional means available for use in conjunction withpharmaceuticals, either as individual therapeutic agents or in acombination of therapeutic agents. They can be administered alone, butgenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice. The compounds can also be administered in conjunction withother prevention or therapeutic agents if desired.

The pharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, or diluents, are well-known to thosewho are skilled in the art. Typically, the pharmaceutically acceptablecarrier is chemically inert to the active compounds and has nodetrimental side effects or toxicity under the conditions of use. Thepharmaceutically acceptable carriers can include polymers and polymermatrices.

The compounds of this disclosure can be administered by any conventionalmethod available for use in conjunction with pharmaceuticals, either asindividual therapeutic agents or in a combination of therapeutic agents.

The dosage administered will, of course, vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent and its mode and route of administration; the age, health andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; and the effectdesired. A daily dosage of active ingredient can be expected to be about0.001 to 1000 milligrams (mg) per kilogram (kg) of body weight, with thepreferred dose being 0.1 to about 30 mg/kg.

Dosage forms (compositions suitable for administration) typicallycontain from about 1 mg to about 500 mg of active ingredient per unit.In these pharmaceutical compositions, the active ingredient willordinarily be present in an amount of about 0.5-95% weight based on thetotal weight of the composition.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, and powders, or in liquid dosage forms, suchas elixirs, syrups and suspensions. It can also be administeredparenterally, in sterile liquid dosage forms. The active ingredient canalso be administered intranasally (nose drops) or by inhalation of adrug powder mist. Other dosage forms are potentially possible such asadministration transdermally, via patch mechanism or ointment.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations may include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, propylene glycol, glycerin, and thepolyethylene alcohols, either with or without the addition of apharmaceutically acceptable surfactant, suspending agent, or emulsifyingagent. Capsule forms can be of the ordinary hard- or soft-shelledgelatin type containing, for example, surfactants, lubricants, and inertfillers, such as lactose, sucrose, calcium phosphate, and corn starch.Tablet forms can include one or more of the following: lactose, sucrose,mannitol, corn starch, potato starch, alginic acid, microcrystallinecellulose, acacia, gelatin, guar gum, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, calcium stearate, zincstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and pharmacologically compatiblecarriers. Lozenge forms can comprise the active ingredient in a flavor,usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin, or sucrose and acadia, emulsions, and gels containing, inaddition to the active ingredient, such carriers as are known in theart.

The compounds of the present disclosure, alone or in combination withother suitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, and nitrogen. They also may beformulated as pharmaceuticals for non-pressured preparations, such as ina nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The compound can be administered in a physiologically acceptable diluentin a pharmaceutical carrier, such as a sterile liquid or mixture ofliquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol, or hexadecylalcohol, glycols, such as propylene glycol or polyethylene glycol suchas poly(ethyleneglycol) 400, glycerol ketals, such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, afatty acid ester or glyceride, or an acetylated fatty acid glyceridewith or without the addition of a pharmaceutically acceptablesurfactant, such as a soap or a detergent, suspending agent, such aspectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters. Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include (a)cationic detergents such as, for example, dimethyldialkylammoniumhalides, and alkylpyridinium halides, (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylene polypropylene copolymers, (d) amphoteric detergents suchas, for example, alkyl β-aminopropionates, and 2-alkylimidazolinequaternary ammonium salts, and (e) mixtures thereof.

The parenteral formulations typically contain from about 0.5% to about25% by weight of the active ingredient in solution. Suitablepreservatives and buffers can be used in such formulations. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5% toabout 15% by weight. Suitable surfactants include polyethylene sorbitanfatty acid esters, such as sorbitan monooleate and the high molecularweight adducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol.

Pharmaceutically acceptable excipients are also well-known to those whoare skilled in the art. The choice of excipient will be determined inpart by the particular compound, as well as by the particular methodused to administer the composition. Accordingly, there is a wide varietyof suitable formulations of the pharmaceutical composition of thepresent disclosure. The following methods and excipients are merelyexemplary and are in no way limiting. The pharmaceutically acceptableexcipients preferably do not interfere with the action of the activeingredients and do not cause adverse side-effects. Suitable carriers andexcipients include solvents such as water, alcohol, and propyleneglycol, solid absorbants and diluents, surface active agents, suspendingagent, tableting binders, lubricants, flavors, and coloring agents.

The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tablets.The requirements for effective pharmaceutical carriers for injectablecompositions are well known to those of ordinary skill in the art. SeePharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia,Pa., Banker and Chalmers, Eds., 238-250 (1982) and ASHP Handbook onInjectable Drugs, Toissel, 4th ed., 622-630 (1986).

Formulations suitable for topical administration include lozengescomprising the active ingredient in a flavor, usually sucrose and acaciaor tragacanth; pastilles comprising the active ingredient in an inertbase, such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier; as well as creams, emulsions, and gels containing, in additionto the active ingredient, such carriers as are known in the art.

Additionally, formulations suitable for rectal administration may bepresented as suppositories by mixing with a variety of bases such asemulsifying bases or water-soluble bases. Formulations suitable forvaginal administration may be presented as pessaries, tampons, creams,gels, pastes, foams, or spray formulas containing, in addition to theactive ingredient, such carriers as are known in the art to beappropriate.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

The dose administered to an animal, particularly a human, in the contextof the present disclosure should be sufficient to affect a therapeuticresponse in the animal over a reasonable time frame. One skilled in theart will recognize that dosage will depend upon a variety of factorsincluding a condition of the animal, the body weight of the animal, aswell as the severity and stage of the condition being treated.

A suitable dose is that which will result in a concentration of theactive agent in a patient which is known to affect the desired response.The preferred dosage is the amount which results in maximum inhibitionof the condition being treated, without unmanageable side effects.

The size of the dose also will be determined by the route, timing andfrequency of administration as well as the existence, nature, and extendof any adverse side effects that might accompany the administration ofthe compound and the desired physiological effect.

Useful pharmaceutical dosage forms for administration of the compoundsaccording to the present disclosure can be illustrated as follows:

Hard Shell Capsules

A large number of unit capsules are prepared by filling standardtwo-piece hard gelatin capsules each with 100 mg of powdered activeingredient, 150 mg of lactose, 50 mg of cellulose and 6 mg of magnesiumstearate.

Soft Gelatin Capsules

A mixture of active ingredient in digestible oil such as soybean oil,cottonseed oil or olive oil is prepared and injected by means of apositive displacement pump into molten gelatin to form soft gelatincapsules containing 100 mg of the active ingredient. The capsules arewashed and dried. The active ingredient can be dissolved in a mixture ofpolyethylene glycol, glycerin and sorbitol to prepare a water misciblemedicine mix.

Tablets

A large number of tablets are prepared by conventional procedures sothat the dosage unit was 100 mg of active ingredient, 0.2 mg. ofcolloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg ofmicrocrystalline cellulose, 11 mg. of starch, and 98.8 mg of lactose.Appropriate aqueous and non-aqueous coatings may be applied to increasepalatability, improve elegance and stability or delay absorption.

Immediate Release Tablets/Capsules

These are solid oral dosage forms made by conventional and novelprocesses. These units are taken orally without water for immediatedissolution and delivery of the medication. The active ingredient ismixed in a liquid containing ingredient such as sugar, gelatin, pectinand sweeteners. These liquids are solidified into solid tablets orcaplets by freeze drying and solid state extraction techniques. The drugcompounds may be compressed with viscoelastic and thermoelastic sugarsand polymers or effervescent components to produce porous matricesintended for immediate release, without the need of water.

Moreover, the compounds of the present disclosure can be administered inthe form of nose drops, or metered dose and a nasal or buccal inhaler.The drug is delivered from a nasal solution as a fine mist or from apowder as an aerosol.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference, and for any and allpurpose, as if each individual publication, patent or patent applicationwere specifically and individually indicated to be incorporated byreference. In the case of inconsistencies, the present disclosure willprevail.

The foregoing description of the disclosure illustrates and describesthe present disclosure. Additionally, the disclosure shows and describesonly the preferred embodiments but, as mentioned above, it is to beunderstood that the disclosure is capable of use in various othercombinations, modifications, and environments and is capable of changesor modifications within the scope of the concept as expressed herein,commensurate with the above teachings and/or the skill or knowledge ofthe relevant art.

The embodiments described hereinabove are further intended to explainbest modes known of practicing it and to enable others skilled in theart to utilize the disclosure in such, or other, embodiments and withthe various modifications required by the particular applications oruses. Accordingly, the description is not intended to limit it to theform disclosed herein. Also, it is intended that the appended claims beconstrued to include alternative embodiments.

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1. A compound represented by the formulae (1) or (2):

wherein in formula (1) R₁ is a hydroxy function or an alkoxy groupcontaining 1-4 carbon atoms; R₂ is a acyl halo group containing 2-5carbon atoms; R₃ is a halo group or an alkoxy group containing 1-4carbon atoms and R₄ is H when R₃ is halo and is an alkoxy groupcontaining 1-4 carbon atoms when R₃ is an alkoxy group containing 1-4carbon atoms; wherein in formula (2), R₁ is a hydroxy or an alkoxy groupcontaining 1-4 carbon atoms; R₂ is an acyl halo group containing 2-3carbon atoms; R₃ is a halo group and R₄ is H or halo; pharmaceuticallyacceptable salt thereof; prodrugs thereof or solvates thereof.
 2. Acompound according to claim 1 being represented by formula 1,pharmaceutically acceptable salt thereof; prodrugs thereof or solvatesthereof.
 3. A compound according to claim 1 being represented by formula2, pharmaceutically acceptable salt thereof; prodrugs thereof orsolvates thereof.
 4. A compound according to claim 1 wherein R₃ ischloro or bromo.
 5. A compound according to claim 1 wherein R₄ isselected from the group consisting of hydrogen, methoxy and chloro.
 6. Acompound according to claim 1 wherein R₂ is ethanoyl chloride.
 7. Acompound according to claim 1 being selected from the group consistingof:methyl-1-(2-bromophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;methyl-1-(2,5-dimethoxyphenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;methyl1-(2,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;methyl1-(3,4dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;and methyl 1-(2,6-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro1H-β-carboline-3-carboxylate; pharmaceutically acceptable salt thereof;prodrugs thereof or solvates thereof.
 8. A compound according to claim 1being selected from the group consisting of: methyl(1R,3R)-1-(2-bromophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;methyl(1S,3R)-1-(2-bromophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;methyl(1S,3S)-1-(2-bromophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;methyl(1R,3S)-1-(2-bromophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;methyl(1R,3R)-1-(2,5-dimethoxyphenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;methyl(1S,3R)-1-(2,5-dimethoxyphenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;methyl(1S,3S)-1-(2,5-dimethoxyphenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;methyl(1R,3S)-1-(2,5-dimethoxyphenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;(1S,3R) methyl1-(2,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;(1S,3S) methyl1-(2,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;(1S,3S) methyl1-(2,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;(1S,3R) methyl1-(3,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;(1S,3R) methyl1-(3,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;(1S,3S) methyl1-(3,4-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;(1R,3S) methyl1-(3,4-dichloro-phenyl)-2(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate;(1S,3R) methyl1-(2,6-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro1H-β-carboline-3-carboxylate; (1S,3S) methyl1-(2,6-dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxyl;pharmaceutically acceptable salt thereof; prodrugs thereof or solvatesthereof.
 9. A pharmaceutical composition containing a compound accordingto claim 1; pharmaceutically acceptable salt thereof; prodrug thereof orsolvate thereof.
 10. A method for treating a mammalian host in need ofan anticancer treatment, which comprises administering to said host aneffective anticancer amount of at least one compound according to claim1; pharmaceutically acceptable salt thereof; prodrugs thereof orsolvates thereof.
 11. A method for treating male erectile dysfunction,which comprises administering to said host an effective amount of atleast one compound according to claim 1; pharmaceutically acceptablesalt thereof; prodrugs thereof or solvates thereof.
 12. A method fortreating pulmonary hypertension, which comprises administering to saidhost an effective amount of at least one compound according to claim 1;pharmaceutically acceptable salt thereof; prodrugs thereof or solvatesthereof.
 13. A method for improving cognitive function which comprisesadministering to said host an effective amount of at least one compoundaccording to claim 1; pharmaceutically acceptable salt thereof; prodrugsthereof or solvates thereof.
 14. A process for preparing a compoundaccording to claim 1 which comprises subjecting a tryptophan ester and asubstituted bromobenzaldehde selected from the group consisting of a2-halobenzaldehyde, 2,5-dialkoxybenzaldehyde, 2,4-dihalobenzaldehyde,3,4-dihalobenzaldehyde and 2,6-dihalobenzaldehdye to a Pictet-Spenglerreaction to thereby obtain 1,3-disubstituted-tetrahydro-β-carbolinecompound and then reacting the 1,3-disubstituted-tetrahydro-β-carbolinecompound with chloroacetyl chloride to provided the desired compoundaccording to claim
 1. 15. The process according to claim 14 wherein cis-and trans-diastereomers of the 1,3-disubstituted-tetrahydro-β-carbolinecompound are formed and which further comprises separating the cis- andtrans-diastereomers of the 1,3-disubstituted-tetrahydro-β-carbolinecompound by column chromatography using CH₂Cl₂ as an eluent.
 16. Apharmaceutical composition containing a compound according to claim 2;pharmaceutically acceptable salt thereof; prodrug thereof or solvatethereof.
 17. A pharmaceutical composition containing a compoundaccording to claim 3; pharmaceutically acceptable salt thereof; prodrugthereof or solvate thereof.
 18. A pharmaceutical composition containinga compound according to claim 4; pharmaceutically acceptable saltthereof; prodrug thereof or solvate thereof.
 19. A pharmaceuticalcomposition containing a compound according to claim 5; pharmaceuticallyacceptable salt thereof; prodrug thereof or solvate thereof.
 20. Apharmaceutical composition containing a compound according to claim 6;pharmaceutically acceptable salt thereof; prodrug thereof or solvatethereof.